1. Field of the Invention
The present invention relates to an attenuated Japanese encephalitis virus adapted to Vero cell by passages on Vero cell and a Japanese encephalitis vaccine comprising said attenuated virus.
2. Description of the Prior Art
Japanese encephalitis (JE) is a mosquito-borne arboviral disease of major or public health importance in Asia. More than 35,000 cases and 10,000 deaths are reported annually from that continent, but official reports undoubtedly underestimate the true number of cases (Okuno, T. World Health Stat Q. 3: 120-31,1978; Umenei, T. et al. Bull World Health Org. 63: 625-31, 1985). The illness may be manifested by a febrile headache syndrome, aseptic meningitis, or encephalitis and about half of the survivors tend to have permanent neurologic and psychiatric sequelae (Burke, D. S. et al. The Arbovirus: Epidemiology and Ecology 3:63-92, 1988; Monath, T. P. Virology 763-814, 1990).
JE virus is one of 66 Flaviviridae, enveloped, positive-sense, single stranded RNA viruses that largely are vector-borne (Chambers, T. J. et al. Ann. Rev. Microbiol. 44:649-88, 1990). Morphologically, flaviviruses are spherical, approximately 40 nm in diameter, are composed of a lipid bilayer surrounding a neucleocapsid containing 11-kb genome complexed with a capsid (C) protein (Rice, C. M. et al. Science 229:726-33, 1985). Surface projections on the membrane are composed of glycosylated envelope (E) and membrane (M) proteins. A pre-M glycoprotein, present in intracellular nascent virions, is cleaved to the M protein, found in mature extracellular virions. Important physiological activities are associated with the 53-kd E protein, including hemagglutination, viral neutralization, virion assembly, membrane fusion, and viral binding to cellular receptors (Koshini, E. et al. Virol. 188:714-20, 1992).
There are three JE vaccines for humans (Tsai, T. et al. Vaccines 671-713, 1993). Of the three, only inactivated JE vaccine produced in mouse brain is available internationally. One manufacturer, the Research Foundation for Microbial Diseases of Osaka University (Biken) produces most of the inactivated JE vaccine distributed internationally; that vaccine was licensed in 1992 in the USA where it is distributed by Connaught Laboratories, Inc., as JE-VAX.TM.. Inactivated and live attenuated JE vaccine prepared in primary hamster kidney (PHK) cells are distributed solely in China.
Inactivated JE vaccine produced in mouse brains was licensed in Japan in 1954. Because it is produced by cerebral injection of infant mice, it is laborious to manufacture and concerns about the possibility of vaccine-related neurological side effect were raised. Though successive refinements in the manufacturing process have increased its purity and potency (Oya, A. Vaccination Theory and Practice 69-82, 1975; Oya, A. Acta Pediatr Jpn. 30:175-84, 1988; Takaku, K. Biken J. 11:25-39, 1968), a moderate frequency of local and mild systemic reactions have been reported until recently (Hoke, C. H. et al. New Engl J Med. 319:608-14, 1988; Poland, J. D. et al. J Infect Dis. 161:878-82, 1990; Sanchez, J. L. et al. Lancet 335:972-73, 1990). Local tenderness, redness, and/or swelling at the injection site occur in 20% of vaccines. Mild systemic symptoms, chiefly headache, low-grade fever, myalgias, malaise, and gastrointestinal symptoms, are reported by 10 to 30% of vaccines. An apparently new pattern of adverse reactions including urticaria, angioedema, respiratory distress, erythema multiforme, erythema nosodum and severe neurological disorders have been reported since 1989, principally among travellers vaccinated in Australia, Europe, and North America (Anderson, M. M. et al. Lancet. 337:1044, 1991; Ruff, T. A. et al. Lancet 228:881-2, 1991). In addition, in 1992 and 1995 Ohtaki reported seven children with acute disseminated encephalomyelitis (ADEM) with changes on magnetic resonance images (MRI) after JE vaccination (Ohtaki, E. et al. Pediatr Neurol. 8:137-9, 1992; Ohtaki, E. et al. J Neurol Neurosurg Psychiatry 59:316-7, 1995). Also of note is that vaccination with rabies vaccine containing animal brain tissue has caused severe neurological complications (Plotkin, S. A. et al. Vaccines 661-2, 1994). For these reasons, the WHO has designated JE vaccine development as a high priority.
More recently, inactivated and live attenuated JE vaccine of China have proven to be effective, eliciting high titers of virus-neutralizing antibody and conferring solid protection (Tsai, T. el al. Vaccines 671-713, 1993). However, PHK cells in which Chinese vaccine were prepared are not approved by the World Health Organization (WHO) for viral vaccine production or licensed for human use by the developed countries. The principal disadvantage in using primary hamster cells for the production of vaccines is the uncertainty with regard to the quality of vaccine. Even if specific pathogen free hamsters are used, animals can unexpectedly become infected, being problematic for vaccine production. Occasionally an infection of this type could be undetected for along time. With these criticisms, further controlled studies of the safety of the vaccine are required to allow confidence regarding its widespread use. Another disadvantage of the vaccine production from primary cells is the low rate of harvest of the virus and high cost without allowing mass production.
In view of the above, there is a need for new JE vaccine which is produced in standard cell lines such as Vero or human diploid cells that have been accepted as human vaccine substrates, with good cost effectiveness. Vero cells are transformed but non-tumorigenic cells derived from monkey kidney. The Vero cell line is more advantageous than any other standard cell line in that Vero cells are more readily adaptable to large scale cell culture and as a transformed cell has an infinite life time.
It has now been found that JE virus can be grown in Vero cell culture. Considerable efforts had been made in the field of JE vaccine to produce vaccine in standard cells which permit effecting cell cultures at a large volume. Nevertheless, virus characterization including genetic stability, yield and process necessary for vaccine commercialization through cultivation with Vero cells had never met the requirements of human vaccine. Owing to these facts and to the difficulties of transposing a knowledge acquired in other virus cultures to JE virus, the prior art had not achieved success in the development of JE virus vaccine which is genetically stable and has a high immunogenic character from continuous cell lines. Among all these researches, none had resulted up to the present time in a new vaccine production which satisfies the criteria mentioned in this background.
The present invention suggests a development and a propagation of JE virus in continuous cell line, Vero cells for vaccine production which overcome previous problems in JE virus produced in mouse brain or primary cell lines. The present invention also identifies methodology developed to cultivate the JE virus and a downstream process for vaccine production with cost-effectiveness.
In addition, the present invention identifies methodology which improves upon the previously commercialized JE vaccines in the following ways.
1. Safety: The invented virus did not acquire the virulence through the Vero cell cultivation, reducing the hazards of production and affording an additional level of safety to recipients beyond that furnished by stringent control over the virus-inactivation process. This advantage has never been provided by the previously commercialized JE vaccines. PA1 2. Increased supply in safer production substrate: The JE vaccine of the present invention is produced in the absence of bovine serum, making high yields and inexpensive and scalable production which are not achieved in the previously commercialized JE vaccines. PA1 3. Less reactogenicity: No gelatin stabilizer is incorporated into the JE vaccine of the present invention, reducing the risk of vaccine reactions like those seen with the existing vaccine (Saskaguchi M. et al. Vaccines 68-69, 1998). In addition, undesirable bovine-derived components, incorporated in the existing JE vaccines are effectively eliminated. Conclusively, this safety point of the present invention has never been provided by the previous JE vaccine. PA1 4. Increased potency: The success of scalable production with Vero cells and the absence of supplements for production, as well as the effective purification, permits the first use of potent adjuvants in formulating the JE vaccine. Although the use of the adjuvants in the vaccine formulation has been applied in other vaccine, transposing this knowledge to the JE vaccine has been in difficulty since none of the existing JE vaccines assure it's safe production.
In conclusion, none had resulted up to the present time in a new vaccine which satisfies the aforementioned advantages in the commercialization of JE vaccine.
Therefore, an object of the present invention is to provide a safe and effective JE vaccine produced in standard cell substrate to increase its acceptability in many countries. A further object of the invention is to provide an effective process for producing a highly purified stable vaccine and formulating a vaccine which has a high immuniogenic character with a small antigen amount.